With the development of genetic recombination technology, a number of proteins have become available for therapeutic use. By way of example, proteins that are currently used to treat diseases include erythropoietin, Factor VIII, Factor IX, hemoglobin, insulin, interferons alpha, beta, and gamma, vascular endothelial growth factor, interleukin 2, and many others. However, proteins can lose biological activity as a result of physical instabilities (e.g., denaturation, formation of aggregates, etc.) and chemical instabilities, such as hydrolysis, oxidation, and deamidation. Stability of proteins is further influenced by factors such as pH, temperature, tonicity, and number of freeze-thaw cycles.
To ensure stability, therapeutic protein formulations are generally supplied either as a lyophilized protein to be dissolved just before use in a separately packaged water-soluble diluent, or as a protein solution containing additives for improving stability. For example, additives such as free amino acids (e.g., leucine, tryptophan, serine, arginine and histidine) useful in formulating protein solutions have been proposed in patents such as, e.g., AU 722300; U.S. Pat. Nos. 5,691,312; 6,120,761. Some protein formulations currently available on the market contain a protein as a stabilizer. For example, human serum albumin or purified gelatin are used to suppress chemical and physical changes in protein solutions. However, the addition of these proteins involves a complicated process for removing viral contamination. Lyophilization is another method used to ensure stability; however, this process increases manufacturing costs, and involves an increased risk of improper administration, as the lyophilized protein needs to be dissolved just prior to the use. U.S. Pat. No. 5,705,482 discloses a pharmaceutical solution of a human growth hormone (hGH) that is stabilized with the peptide Leu-His-Leu.
One of the proteins widely used as a therapeutic agent is erythropoietin. Erythropoietin is a 34–39 kDa glycoprotein hormone that stimulates the formation of red blood cells. It is produced in the kidney, and once produced, it circulates to the bone marrow where it stimulates the conversion of primitive precursor cells into proerythroblasts which subsequently mature into red blood cells. In the normal healthy state, erythropoietin is present in very low concentrations in plasma, i.e., about 0.01 to 0.03 U/ml, but when hypoxia occurs, i.e., the level of oxygen in transport is reduced, the kidney produces more erythropoietin. Hypoxia can be the result of e.g., the loss of large amounts of blood, destruction of red blood cells by radiation, or exposure to high altitudes. In addition, various forms of anemia cause hypoxia since red blood cells are responsible for oxygen transport in the body. In the normal state, an increased level of erythropoietin stimulates the production of new red blood cells thereby raising the level of oxygen and reducing or eliminating the hypoxic condition.
In contrast to this correction of hypoxia which occurs normally, patients with chronic renal failure (“CRF”) have limited or no production of erythropoietin, and consequently, do not produce sufficient red blood cells. As the normal life span for red blood cells is about 120 days, such patients become increasing anemic with time. Prior to the development of recombinant erythropoietin, patients with chronic renal failure often had to undergo regular blood transfusions to maintain a minimum level of red blood cells.
There are several forms of erythropoietin currently used to treat patients—erythropoietin alpha, beta, omega, and delta. Erythropoietin omega is a recombinant protein expressed from the Apal fragment of human genomic erythropoietin DNA transformed into baby hamster kidney (BHK) cells. Erythropoietin omega and its expression are described in, e.g., U.S. Pat. No. 5,688,679. Furthermore, the structure and composition of carbohydrate residues in EPO omega has been described, e.g., in Nimtz et al., (Eur. J. Biochem., 213:39, 1993) and Tsuda et al., (Eur. J. Biochem., 188:405, 1990). EPO omega has an average molecular weight of about 35 kDa and is comprised of multiple isoforms (e.g., by isoelectric focusing, about 6–8 isoforms in broad cut fractions and 6 isoforms in peak fractions)), which is indicative of differing types and amounts of glycosylation, and in particular, different amounts of sialylation. EPO omega has an O-linked oligosaccharide content of less than 1 mole per mole of glycoprotein and has three N-glycosylation sites at amino acid residues Asn-24, Asn-38, and Asn-83 and an O-glycosylation site at amino acid residue Ser-126. Furthermore, unlike urinary human erythropoietin or erythropoietin alpha or beta, EPO omega retains substantially all of its in vivo biological activity even after being subjected to conditions that lead to substantial, if not complete N-deglycosylation, as reported in Sytkowski et al. (Biochem. Biophys. Res. Commun., 176(2):698–704, 1991).
Although commercially available EPO formulations are generally well tolerated and stable, under extreme conditions, such formulations may be unstable and undergo activity losses. These activity losses can be attributed to a destruction of the EPO by catalytic effects of the surface of the ampule used for storage due to traces of heavy metals, atmospheric oxygen and the like, and also, to a deposition of EPO molecules on the vessel wall, a partial denaturing thereof possibly also taking place. Considering the fact that only a few micrograms are present in each dosage unit, the losses due to adsorption can be considerable, even after a short storage time. Furthermore, activity losses may be accelerated by external factors such as heat and light, or in formulations that are free of human blood products such as albumin, or in multi-dose formulations which contain preservatives such as benzyl alcohol.
Accordingly, pharmaceutical protein formulations, which exhibit improved stability without the need to lyophilize a desired protein or use human proteins as stabilizers are desirable.